Metal-based formulations with high microbicidal activity.

Substances were evaluated for their relative potencies in inactivating Junin virus, Escherichia coli, and spores of Bacillus subtilis. Under the conditions of our test, glutaraldehyde was the most efficient agent among all substances currently recommended for disinfecting and sterilizing medical devices. Either copper or iron ions by themselves were able to inactivate virus with an efficiency similar to that of substances currently used for disinfection and sterilization. The microbicidal effect of metals, however, was enhanced further by the addition of peroxide. The mixtures of copper and peroxide described here were more efficient than glutaraldehyde in inactivating viruses and bacteria. The addition of a metal chelator to metal-peroxide mixtures further increased the microbicidal potency of the reagent. The formulations described in this study should be harmless to people but able to quickly and efficiently inactivate microorganisms, particularly viruses.     

Virus inactivation by copper or iron ions alone and in the presence of peroxide.

Cupric and ferric ions were able to inactivate five enveloped or nonenveloped, single- or double-stranded DNA or RNA viruses. The virucidal effect of these metals was enhanced by the addition of peroxide, particularly for copper(II). Under the conditions of our test, mixtures of copper(II) ions and peroxide were more efficient than glutaraldehyde in inactivating phi X174, T7, phi 6, Junin, and herpes simplex viruses. The substances described here should be able to inactivate most, if not all, viruses that have been found contaminating medical devices.     

Research on substances inactivating the X virus present in sap from potato leaves

The work ofJermoljev andBr?ák (1964) showed that the sap of potato leaves contains substances inactivating X, Y and S viruses and that there was an increasing trend in the content of these substances in varieties resistant to these viruses. In further research it was found that potato tubers of varieties which are resistant and susceptible to the viruses contained the same amounts of inactivating substances. Differences in the power of inactivating the viruses evidently appeared in the leaves. Inactivating substances could be removed by boiling, they did not pass through a dialysing membrane and were adsorbed by animal charcoal. On centrifuging sap from potato leaves in a Spinco L ultracentrifuge for 60 min. at 40,000 r.p.m., the inactivating substances remained in the supernatant. When sucrose gradient centrifugation for 60 min. at 24,000 r.p.m. was employed, the inactivating substances remained in the layer of sap and 10% sucrose. Inhibition of the activity of certain enzyme groups did not affect the power of sap to inactivate X virus. Inactivating substances could be isolated chemically. The best method of isolation, however, was fractionation of sap, after ultracentrifugation, on a Sephadex G 50 column, rinsing the column with McIlvaine buffer at pH 6·5. Inactivating substances can be isolated, concentrated and preserved by lyophilization by this method. It is difficult to say precisely to what chemical group the inactivating substances belong. Some reactions indicate that they may be low molecular proteins.     

Microbial inactivation for safe and rapid diagnostics of infectious samples

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log(10)) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseudomonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost.     

Direct inactivation of viruses by human granulocyte defensins.

Human neutrophils contain a family of microbicidal peptides known as defensins. One of these defensins, human neutrophil peptide (HNP)-1, was purified, and its ability to directly inactivate several viruses was extensively tested. Herpes simplex virus (HSV) types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN were inactivated by incubation with HNP-1. Two nonenveloped viruses, echovirus type 11 and reovirus type 3, were resistant to inactivation. Purified homologous peptides HNP-2 and HNP-3 were found to have HSV-1-neutralizing activities approximately equal to that of HNP-1. Inactivation of HSV-1 by HNP-1 depended on the time, temperature, and pH of incubation. Antiviral activity was abrogated by low temperature or prior reduction and alkylation of the defensins. Addition of serum or serum albumin to the incubation mixtures inhibited neutralization of HSV-1 by HNP-1. We used density gradient sedimentation techniques to demonstrate that HNP-1 bound to HSV-1 in a temperature-dependent manner. We speculate that binding of defensin peptides to certain viruses may impair their ability to infect cells.     

Chemical disinfection of high-consequence transboundary animal disease viruses on nonporous surfaces

Disinfection is a critical part of the response to transboundary animal disease virus (TADV) outbreaks by inactivating viruses on fomites to help control infection. To model the inactivation of TADV on fomites, we tested selected chemicals to inactivate Foot and Mouth Disease virus (FMDV), African Swine Fever virus (ASFV), and Classical Swine Fever virus (CSFV) dried on steel and plastic surfaces. For each of these viruses, we observed a 2 to 3 log reduction of infectivity due to drying alone. We applied a modified surface disinfection method to determine the efficacy of selected disinfectants to inactivate surface-dried high-titer stocks of these three structurally different TADV. ASFV and FMDV were susceptible to sodium hypochlorite (500 and 1000 ppm, respectively) and citric acid (1%) resulting in complete disinfection. Sodium carbonate (4%), while able to reduce FMDV infectivity by greater than 4-log units, only reduced ASFV by 3 logs. Citric acid (2%) did not totally inactivate dried CSFV, suggesting it may not be completely effective for disinfection in the field. Based on these data we recommend disinfectants be formulated with a minimum of 1000 ppm sodium hypochlorite for ASFV and CSFV disinfection, and a minimum of 1% citric acid for FMDV disinfection.     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Potentially Infectious Agents Associated with Shearling Bedpads: I. Effect of Laundering with Detergent-Disinfectant Combinations on Polio and Vaccinia Viruses

Glutaraldehyde-tanned woolskin pads which are used for the prevention of decubitus ulcers in bed patients were experimentally contaminated with polio or vaccinia viruses. Two methods of exposure, direct contact and aerosol, were used in separate experiments. Attempts were made to remove or inactivate these virus contaminants by laundering the woolskins in a quaternary ammonium disinfectant, a phenolic disinfectant, or alkalinized glutaraldehyde, in combination with an anionic detergent or a nonionic detergent. The effect of a commercial detergent-sanitizer was also studied. The virus titers were significantly reduced in all experiments, but only laundering in glutaraldehyde in combination with either detergent lowered the vaccinia virus titers to below detectable limits. High concentrations of glutaraldehyde altered the texture of the wool and leather apparently by precipitating a component of the detergent onto the fibers. In all the poliovirus experiments, the virus was still detectable on either or both the wool and the leather of the pads after laundering. The rinse water from each experiment was tested for the presence of virus. No vaccinia virus was recovered, but poliovirus was demonstrated in titers up to 10(3) cell culture 50% infectious doses.     

Novel amphiphilic compounds effectively inactivate the vaccinia virus

Recent studies demonstrated the ability of artificial ribonucleases (aRNases, small organic RNA cleaving compounds) to inactivate RNA-viruses via the synergetic effect of viral RNA cleavage and disruption of viral envelope [1,2]. Herein, we describe the antiviral activity of aRNases against DNA-containing vaccinia virus: screening of aRNases of various structures revealed that amphiphilic compounds built of positively charged 1,4-diazabicyclo[2.2.2] octane substituted at the bridge nitrogen atoms with aliphatic residues efficiently inactivate this virus. The first stage was the destruction of viral membrane and structure of surface proteins (electron microscopy data). Thus, 1,4-diazabicyclo[2.2.2] octane-based aRNases are novel universal agents inactivating both RNA- and DNA-containing viruses.     

Septage treatments to reduce the numbers of bacteria and polioviruses.

Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended.     

Septage treatments to reduce the numbers of bacteria and polioviruses

Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended.     

Chemical inactivation of HIV on surfaces.

To assess whether alcohol and glutaraldehyde are effective disinfectants against dried HIV the virucidal effects of 70% alcohol (ethanol and industrial methylated spirit) and 1% and 2% alkaline glutaraldehyde were tested against cell associated and cell free HIV dried on to a surface. Virus stock (100 microliters) or 10,000 cultured C8166 T lymphocytes infected with HIV were dried onto sterile coverslips and immersed in 2% and 1% alkaline glutaraldehyde and 70% ethanol for 30 seconds and one, two, four, and 10 minutes, there being an additional time point of 20 minutes for cell free virus disinfected with 70% industrial methylated spirit. In addition, virus stock in neat serum was tested with 1% and 2% alkaline glutaraldehyde to see whether the fixative properties of glutaraldehyde impair its virucidal properties. Virus activity after disinfection was tested by incubating the coverslips (cell associated virus) or the coverslips and sonicated cell free virus with C8166 T lymphocytes. The lymphocytes were examined for the formation of syncytia and HIV antigens were assayed in the culture fluid. Both 2% and 1% alkaline glutaraldehyde inactivated cell free HIV within one minute; 2% alkaline glutaraldehyde also inactivated cell free virus in serum within two minutes, but a 1% solution was ineffective after 15 minutes'' immersion. Cell associated HIV was inactivated by 2% alkaline glutaraldehyde within two minutes. Seventy per cent industrial methylated spirit failed to inactivate cell free and cell associated HIV within 20 and 15 minutes, respectively, and 70% ethanol did not inactivate cell free virus within 10 minutes. Seventy per cent industrial methylated spirit and ethanol are not suitable for surface disinfection of HIV. Fresh 2% solutions of alkaline glutaraldehyde are effective, but care should be taken that they are not too dilute or have not become stale when used for disinfecting HIV associated with organic matter.     

一种新型网膜对SARS冠状病毒的抑制作用

利用光触媒钛羟基磷灰石网膜（PTAF）具有吸附和酶催化的特点,研究其对SARS病毒的抑制作用.实验结果表明在紫外照射条件下,PTAF膜对SARS冠状病毒的抑制率为100％.在没有紫外照射的条件下,PTAF膜对SARS冠状病毒的抑制率为99.99％, 与对照组相比,PTAF膜抑制病毒的效率是HAF膜的1 000倍以上.研究结果提示,PTAF在预防SARS冠状病毒及其他病毒性疾病流行方面有潜在的应用价值.     

Enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates.

Solvent-detergent treatment, although used routinely in plasma product processing to inactivate enveloped viruses, substantially reduces product yield from the human plasma resource. To improve yields in plasma product manufacturing, a new viral reduction process has been developed using the fatty acid caprylate. As licensure of plasma products warrants thorough evaluation of pathogen reduction capabilities, the present study examined susceptibility of enveloped viruses to inactivation by caprylate in protein solutions with varied pH and temperature. In the immunoglobin-rich solutions from Cohn Fraction II+III, human immunodeficiency virus, Type-1, bovine viral diarrhea virus (BVDV), and pseudorabies virus were inactivated by caprylate concentrations of >/=9 mM, >/=12 mM, and >/=9 mM, respectively. Compared to solvent-detergent treatment, BVDV inactivation in Fraction II+III solution was significantly faster (20-60 fold) using 16 mM caprylate. Caprylate-mediated inactivation of BVDV was not noticeably affected by temperature within the range chosen manufacturing the immunoglobulin product. In Fraction II+III solutions, IgG solubility was unaffected by 相似文献   

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Improving baculovirus recombination

Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation.     

Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility.

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.     

Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility

A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.     

Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus

Gene delivery vectors based on adeno-associated virus (AAV) have significant therapeutic potential, but much room for improvement remains in the areas of vector engineering and production. AAV production requires complementation with either helper virus, such as adenovirus, or plasmids containing helper genes, and helper virus-based approaches have distinct advantages in the use of bioreactors to produce large quantities of AAV vectors for clinical applications. However, helper viruses must eventually be inactivated and removed from AAV preparations to ensure safety. The current practice of thermally inactivating adenovirus is problematic as it can also inactivate AAV. Here, we report a novel method using high hydrostatic pressure (HHP) to selectively and completely inactivate helper adenovirus without any detectable loss of functional AAV vectors. The pressure inactivation kinetics of human adenovirus serotype 5 and the high-pressure stabilities of AAV serotypes 2 and 5 (AAV2, AAV5), which were previously unknown, were characterized. Adenovirus was inactivated beyond detection at 260 MPa or higher, whereas AAV2 was stable up to approximately 450 MPa, and surprisingly, AAV5 was stable up to at least 700 MPa. The viral genomic DNA of pressure-inactivated AAV2 was made sensitive to DNAse I digestion, suggesting that gross changes in particle structure had occurred, and this hypothesis was further supported by transmission electron microscopy. This approach should be useful in the laboratory- and clinical-scale production of AAV gene delivery vectors. Moreover, HHP provides a tool for probing the biophysical properties of AAV, which may facilitate understanding and improving the functions of this important virus.     

Ammonia disinfection of hatchery waste for elimination of single-stranded RNA viruses

Hatchery waste, an animal by-product of the poultry industry, needs sanitation treatment before further use as fertilizer or as a substrate in biogas or composting plants, owing to the potential presence of opportunistic pathogens, including zoonotic viruses. Effective sanitation is also important in viral epizootic outbreaks and as a routine, ensuring high hygiene standards on farms. This study examined the use of ammonia at different concentrations and temperatures to disinfect hatchery waste. Inactivation kinetics of high-pathogenic avian influenza virus H7N1 and low-pathogenic avian influenza virus H5N3, as representatives of notifiable avian viral diseases, were determined in spiked hatchery waste. Bovine parainfluenza virus type 3, feline coronavirus, and feline calicivirus were used as models for other important avian pathogens, such as Newcastle disease virus, infectious bronchitis virus, and avian hepatitis E virus. Bacteriophage MS2 was also monitored as a stable indicator. Coronavirus was the most sensitive virus, with decimal reduction (D) values of 1.2 and 0.63 h after addition of 0.5% (wt/wt) ammonia at 14 and 25°C, respectively. Under similar conditions, high-pathogenic avian influenza H7N1 was the most resistant, with D values of 3.0 and 1.4 h. MS2 was more resistant than the viruses to all treatments and proved to be a suitable indicator of viral inactivation. The results indicate that ammonia treatment of hatchery waste is efficient in inactivating enveloped and naked single-stranded RNA viruses. Based on the D values and confidence intervals obtained, guidelines for treatment were proposed, and one was successfully validated at full scale at a hatchery, with MS2 added to hatchery waste.